Conus snails synthesize a bewildering variety of oligopeptides which target neuronal receptors and ion channels. The peptides contain an invariant framework of disulfides, while the remaining amino acid residues are relatively random and contain a gamut of post-translational modifications which are essential for their biological activity. Conus have evolved an efficient genetic means for encoding the variations. One of our goals is to understand how this diversity is generated. This will be achieved by determining the structure and sequence of the known conotoxin genes and their cDNAs. We will also attempt to characterize additional conotoxins by sequencing of selected cDNAs obtained from venom duct after elimination by subtractive hybridization of those that have been previously identified. We hope this knowledge will enable use to design efficient combinatorial methods for obtaining novel ligands of pharmacological import. A second goal of the project is to understand the mechanism of post- translational modification of the conotoxins, especially how gamma- carboxylation of glutamate is achieved. This modification is found in some conotoxins and in all conantokins, and is essential to their activity. Conus are the only non-mammalian species known to elaborate this modification. In mammals, gamma-carboxylated proteins are vital for the homeostasis of the blood coagulation cascade. Our aim is identify and characterize the enzymes necessary for gamma-carboxylation. This will be done by biochemical isolation of the enzyme and cloning of the gene. We hope the Conus gamma-carboxylation system is sufficiently similar to the mammalian system, and will also afford insight for the synthesis of active coagulation factors.